universiti putra malaysia rat cytomegalovirus …psasir.upm.edu.my/id/eprint/42963/1/fpv 2013 13...
TRANSCRIPT
UNIVERSITI PUTRA MALAYSIA
RAT CYTOMEGALOVIRUS GENOME SCAFFOLD AND A033 GENE AS INFECTION MARKER IN RATS
SITI NAZRINA CAMALXAMAN
FPV 2013 13
© COPYRIG
HT UPM
RAT CYTOMEGALOVIRUS GENOME SCAFFOLD AND
A033 GENE AS INFECTION MARKER IN RATS
By
SITI NAZRINA CAMALXAMAN
Thesis submitted to the School of Graduate Studies, Universiti Putra
Malaysia, in Fulfilment of the Requirements for the Degree of Doctor of
Philosophy
May 2013
© COPYRIG
HT UPM
COPYRIGHT PAGE
All material contained within the thesis, including without limitation text, logos,
icons, photographs and all other artwork, is copyright material of
Universiti Putra Malaysia unless otherwise stated. Use may be made of any material
contained within the thesis for non-commercial purposes from the copyright holder.
Commercial use of material may only be made with the express, prior, written
permission of Universiti Putra Malaysia.
Copyright © Universiti Putra Malaysia
© COPYRIG
HT UPM
DEDICATIONS
Dedicated with great love and extreme gratitude to
My parents:
Camalxaman & Che Nu
My husband:
Adi Suria
My children:
Adi Irfan & Adi Reza
My siblings:
Nazri, Nazrul, Nazmi & Nastiti
“The love of a family is life's greatest blessing”
© COPYRIG
HT UPM
ii
Abstract of thesis presented to the Senate of Universiti Putra Malaysia
in fulfilment of the requirement for the degree of Doctor of Philosophy
RAT CYTOMEGALOVIRUS GENOME SCAFFOLD AND
A033 GENE AS INFECTION MARKER IN RATS
By
SITI NAZRINA CAMALXAMAN
May 2013
Chair: Associate Professor Zeenathul Nazariah Allaudin, PhD.
Faculty: Veterinary Medicine
A local preliminary study has documented the prevalence of rat cytomegalovirus
(RCMV) in the wild population to be as high as 96%. Hence, periodic screening and
surveillance of laboratory rats is vital, since they may also harbor the viral agent,
posing challenges for experimental usage. The lack of sequence information in
RCMV ALL-03 strain however, has impeded its detection and prevented its
assesment in vitro. This thesis describes the reactivation of RCMV ALL-03 from
predilected sites, the establishment of rat brain endothelial cells (RBEC) as
alternative target cells for viral replication and the identification of cross-reactive
viral proteins with human CMV (HCMV). In addition, the draft genome for RCMV
ALL-03 was generated using Next Generation Sequencing technology and assembled
using CLC Genomics Workbench. This has led to the identification, analysis and
primer design for the A033 gene, an infection determinant in RCMV.
RCMV ALL-03 was reactivated from the brain, salivary gland and uterus of infected
tissues and identified based on morphologic criteria classical of herpesvirus. RBEC
primary cells were successfully established and deemed receptive for RCMV ALL-
03 with concomitant production of plaques following cytopathogenic studies.
© COPYRIG
HT UPM
iii
Preliminary serological screening of HCMV in Selangor and Kuala Lumpur revealed
92% endemicity. Protein profiles of RCMV ALL-03 were compared to a local
RCMV strain (RCMV UPM/Sg) and RCMV-E (Rat2; ATCC CRL-1764™)
reference strain, revealing eight common protein bands in the range of 44-231 kDa.
The detection of a 61-68 kDa cross reactive protein by Western Blot raises the
possibility of an immunological cross-reactivity between RCMV and HCMV. The
RCMV ALL-03 draft genome was sequenced alongside RCMV-E, generating six
contigs for RCMV ALL-03 and 11 contigs for RCMV-E. The sizes of RCMV
ALL-03 and RCMV-E draft genome sequences were ~198,895 bp and ~175,071 bp
respectively, with a total of 136 genes for RCMV ALL-03 as opposed to 112 genes
for RCMV-E. From this, only 46 genes were annotated for RCMV ALL-03 and 43
genes in RCMV-E. This includes the A033 gene, identified as gene 21 (1,173 bp) in
RCMV ALL-03 and gene 20 (1,187 bp) in RCMV-E. Specific primer for the A033
gene, a marker for RCMV infection has been proposed, and its specificity validated
using PCR against other viral strains. To conclude, this study confirms that RCMV
ALL-03 is endotheliotropic, justifying its use as an alternative cell culture system
that could be further exploited to study the effects of cellular activation of RCMV in
the brain. Furthermore, this study addresses the lack of sequence information in
RCMV ALL-03, by reporting the first draft of the genome, providing new genomic
data acquisition which has not been disclosed to date. Primers for the A033 gene is
being proposed to replace existing ones that were rendered non-specific, paving way
towards the establishment of a more accurate and sensitive detection assay to screen
for RCMV. Once completed, the genome sequence could be further developed as a
recombinant vector for delivering human chimeric or antifertility genes.
© COPYRIG
HT UPM
iv
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan untuk ijazah Doktor Falsafah
SKAFOLD GENOM SITOMEGALOVIRUS TIKUS DAN
GEN A033 SEBAGAI PENANDA JANGKITAN DALAM TIKUS
Oleh
SITI NAZRINA CAMALXAMAN
Mei 2013
Pengerusi: Profesor Madya Zeenathul Nazariah Allaudin, PhD.
Fakulti: Perubatan Veterinar
Laporan saringan sitomegalovirus tikus (RCMV) sebelum ini menunjukkan 96%
prevalensi dalam populasi liar. Oleh itu, pemeriksaan dan pengawasan tikus makmal
secara berkala adalah penting, kerana virus tersebut boleh juga didapati dalam
tikus-tikus ini, menimbulkan cabaran untuk kegunaan eksperimen. Kekurangan
jujukan data dalam RCMV ALL-03 banyak menghalang pengesanannya secara
in vitro. Tesis ini menerangkan pengaktifan semula RCMV ALL-03 dari kawasan
tertentu, pengasingan sel-sel endothelial otak tikus (RBEC) sebagai sel-sel sasaran
alternatif untuk replikasi virus dan pengenalpastian protein yang terlibat dalam
tindak balas-silang dengan sitomegalovirus manusia (HCMV). Di samping itu, draf
genom untuk RCMV ALL-03 telah dijana menggunakan teknologi Next Generation
Sequencing dan disusun menggunakan CLC Genomic Workbench. Hasil susunan
genom membawa kepada pengenalpastian, analisis dan penghasilan primer bagi gen
A033, sejenis penentu jangkitan dalam RCMV. RCMV ALL-03 telah diaktifkan
semula dari tisu yang telah dijangkiti iaitu tisu otak, kelenjar air liur dan uterus dan
dikenalpasti berdasarkan ciri-ciri morfologi klasik herpesvirus. Sel primer RBEC
telah berjaya diasingkan dan didapati sesuai bagi pembiakkan RCMV ALL-03
© COPYRIG
HT UPM
v
melalui penghasilan plak sejurus selepas kajian sitopatogenik. Saringan serologi awal
HCMV di Selangor dan Kuala Lumpur menunjukkan 92% endemisiti. Perbandingan
profil protein RCMV ALL-03 dengan strain tempatan (RCMV UPM/Sg) dan strain
rujukan RCMV-E (Rat2; ATCC CRL-1764™) menunjukkan lapan jalur protein yang
sama dalam julat 44-231 kDa. Pengenalpastian protein yang terlibat dalam tindak
balas-silang RCMV dan HCMV telah dilaksanakan melalui pengesanan protein
reaktif bersaiz 61-68 kDa menggunakan kaedah Western Blot. Genom RCMV ALL-
03 telah disusun bersama strain RCMV-E menghasilkan enam contigs untuk RCMV
ALL-03 dan 11 contigs untuk RCMV-E. Saiz pencirian jujukan genom RCMV ALL-
03 dan RCMV-E separa adalah masing-masing ~198,895 bp dan ~175,071 bp,
dengan jumlah 136 gen untuk RCMV ALL-03 berbanding 112 gen untuk RCMV-E.
Dari jumlah ini, hanya 46 gen beranotasi untuk RCMV ALL-03 dan 43 gen untuk
RCMV-E. Ini termasuk gen A033, yang telah dikenalpasti sebagai gen 21 (1,173 bp)
dalam RCMV ALL-03 dan gen 20 (1187 bp) dalam RCMV-E. Primer khusus untuk
gen A033, telah direka dan spesifikasinya disahkan dengan kaedah PCR
menggunakan strain virus yang berlainan. Kesimpulannya, kajian ini mengesahkan
bahawa RCMV ALL-03 adalah endoteliotropik. Penemuan ini menunjukkan
kewajaran penggunaan RBEC sebagai sistem kultur sel alternatif yang berpotensi
untuk kajian kesan pengaktifan selular RCMV dalam otak. Tambahan pula, kajian ini
menangani kekurangan maklumat jujukan dalam RCMV ALL-03, dengan
melaporkan draf pertama genom, secara langsung menghasilkan data baru genomik
yang belum pernah didedahkan sehingga hari ini. Primer khusus untuk gen A033
dijangka dapat digunakan untuk menghasilkan asai pengesanan untuk RCMV.
Setelah selesai, jujukan genom seterusnya boleh dijadikan sebagai vektor
rekombinan untuk menyampaikan gen kimera manusia atau gen antifertiliti.
© COPYRIG
HT UPM
vi
ACKNOWLEDGEMENTS
In the name of Allah, the Most Gracious and the Most Merciful.
Syukur Alhamdulillah, all praises to Allah, for granting me the strength, courage and
blessings to conclude the writing of my thesis. I would not have accomplished this
without the help and support from so many people. Firstly, I must acknowledge my
sincere gratitude to my supervisor Assoc. Prof. Dr. Zeenathul Nazariah Allaudin.
Thank you for your guidance, supervision and assistance throughout these years.
I have learnt a great deal from you and for that I am forever indebted. My sincere
appreciation also goes to my co-supervisors: Assoc. Prof. Dr. Zuridah (my dearest
mentor), Assoc. Prof. Dr. Sandy Loh Hwei San, and Prof. Dato’ Dr. Sheikh Omar for
all your constructive suggestions, valuable guidance and fruitful discussions that
have aided my study.
To Yi Wan, you are the best partner one could ever have. Thank you for dividing the
task and multiplying the success. To the rest of Dr Zeenat’s group members: Noraini,
Hidayah, Morvarid, Homayoun, Ruzila, Tan, Nik, Lo, Tam, Caryn, Parisa, Faezah,
Fatemah, Noushin and Tarlan, thank you for making my challenges less challenging,
and my problems less problematic. To Iffah, Fahmi, En. Din, En. Nazri, and Miza,
thank you for sharing your technical expertise and for teaching me from scratch. To
my best friend Dr. Amer and virology lab mates Dr. Mayada, Dr. Saeed, Dr. Faruku,
Dr. Rohaya, Afzal and Dr. Majed, thank you for the warmth of your friendship.
Thank you to UiTM and MOHE for granting me the scholarship and the opportunity
to pursue my dreams in becoming a better educator. To my colleagues: En. Zed,
Ann Erynna, Dr. Emida, Hisham, Nisa, Hadi, Dr. Maimunah, Dr. Mazlina, Dr. Jack,
Pn. Azlin, Hartini, Dr. Mazura, Dr. Izham, Dr. Roslinah and Tn Hj Nazri thank you
for the uncountable support and motivation during the hard and stressful moments.
© COPYRIG
HT UPM
vii
To my beloved parents Camalxaman Md Nor & Che Nu Hashim: thank you for
helping me in all ways imaginable, for praying for me day and night, and for taking
care of the kids while I was busy taking care of my virus. I am truly blessed. To my
parents-in-law Jamaludin Mohamad & Zaini Badiuzzaman, siblings Nazri, Nazrul
Nazmi & Nastiti and extended family, Mazuin, Iya, Elia, Aca, Ainur, Ayu, Sarah and
Wonder, thank you for your love and support. To my soul mate Adi Suria, thank
you for being with me throughout this roller-coaster ride and for being my pillar of
strength during my most difficult times. Without you, I would have crumbled and
I’m certain this work would have never occurred. To Irfan & Reza, you are both the
reasons why I kept on going and never gave up. I apologize for spending more time
in the lab than I did at home, but I promise to catch up on lost time. I hope that my
struggles will inspire you both to reach your fullest potential and to fight for your
dreams.
Last but not least, thank you to the members of my Thesis Examination Committee
who took their time to go through, deliberate and examine my thesis. At last, my
hard work, perseverance and tenacity paid off, and for that I thank Allah.
© COPYRIG
HT UPM
viii
I certify that a Thesis Examination Committee has met on 10 May 2013 to conduct
the final examination of Siti Nazrina Camalxaman on her thesis entitled
"Rat Cytomegalovirus Genome Scaffold and A033 Gene as Infection Marker in
Rats" in accordance with the Universities and University Colleges Act 1971 and the
Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The
Committee recommends that the student be awarded the Doctor of Philosophy.
Members of the Thesis Examination Committee were as follows:
Noordin Mohamed Mustapha, PhD
Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Chairman)
Hassan Hj. Mohd. Daud, PhD
Associate Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Internal Examiner)
Abdul Rahman Omar, PhD
Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Internal Examiner)
Jimmy Kwang, PhD
Professor
National University of Singapore
Singapore
(External examiner)
NORITAH OMAR, PhD
Assoc. Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date: 2 August 2013
© COPYRIG
HT UPM
ix
This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfillment of the requirement for the degree of Doctor of Philosphy.
The members of the Supervisory Committee were as follows:
Zeenathul Nazariah Allaudin, PhD
Associate Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Chairman)
Dato’ Sheikh Omar Abdul Rahman, PhD
Professor
Faculty of Veterinary Medicine
Universiti Putra Malaysia
(Member)
Loh Hwei San, PhD
Associate Professor
Faculty of Sciences
The University of Nottingham Malaysia Campus
(Member)
Zuridah Hassan, PhD
Associate Professor
Faculty of Health Sciences
Universiti Teknologi MARA
(Member)
____________________________
BUJANG BIN KIM HUAT, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date:
© COPYRIG
HT UPM
x
DECLARATION
I declare that the thesis is my original work except for quotations and citations which
have been duly acknowledged. I also declare that it has not been previously, and is
not concurrently submitted for any other degree at Universiti Putra Malaysia or at
any other institutions.
_____________________________
SITI NAZRINA CAMALXAMAN
Date:
© COPYRIG
HT UPM
xi
TABLE OF CONTENTS
Page
ABSTRACT ii
ABSTRAK iv
ACKNOWLEDGEMENTS vi
APPROVAL viii
DECLARATION x
LIST OF TABLES xvi
LIST OF FIGURES xviii
LIST OF ABBREVIATIONS xxi
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW
2.1 General Information 5
2.1.1 Herpesvirus Overview 5
2.1.2 Cytomegaloviruses (CMV) 6
2.1.3 Genome Organization 6
2.1.4 CMV Structure 9
2.1.4.1 Viral Genome 9
2.1.4.2 Nucleocapsid 10
2.1.4.3 Tegument 10
2.1.4.4 Envelope and Glycoproteins 11
2.1.5 CMV Replication Cycle 12
2.1.5.1 Entry and Pre-replication Events 13
2.1.5.2 DNA Replication and Gene
Expression
13
2.1.5.3 Assembly and Egress 15
2.2 Human Cytomegalovirus (HCMV) 17
2.2.1 Brief History 17
2.2.2 Clinical Infection 18
2.2.3 Transmission and Spread 20
2.2.4 Latency and Reactivation 20
2.2.5 Cellular Tropism 23
2.2.6 Laboratory Diagnosis 25
2.2.7 Treatment of Infections 26
2.3 Animal Models for HCMV 28
2.3.1 Rat Cytomegalovirus (RCMV) 29
2.3.2 R33 and G Protein Coupled Receptors 31
2.4 Sequencing Technologies 32
2.4.1 Next Generation Sequencing (NGS) 33
2.4.2 Illumina Sequencing Technology 36
2.4.3 Genome Informatics of the NGS Data 39
2.5 Progress of CMV Vaccine Development 41
© COPYRIG
HT UPM
xii
3 RECOVERY AND REACTIVATION OF RCMV ALL-03
FROM INFECTED RAT
3.1 Introduction 46
3.2 Materials and Methods 49
3.2.1 Cell Culture Preparation 49
3.2.2 Subculture of Confluent REF Cells 49
3.2.3 Virus Reactivation from Infected Tissues 50
3.2.4 Mycoplasma Screening 51
3.2.5 Propagation of Virus 51
3.2.6 Harvesting of Virus 52
3.2.7 Concentration of Virus 52
3.2.8 Purification of Virus 52
3.2.9 Titration of Virus by TCID50 53
3.2.10 Host Range Specificity Studies 54
3.2.11 Negative Contrast Electron Microscopy
(NCEM)
55
3.3 Results 56
3.3.1 Mycoplasma Screening 56
3.3.2 Virus Recovery from Infected Rat Tissues 56
3.3.3 Virus Purification 58
3.3.4 Titration of Virus by TCID50 59
3.3.5 Host Range Specificity Studies 59
3.3.6 NCEM 60
3.4 Discussion 62
3.5 Conclusions 64
4 ESTABLISHMENT AND INFECTIVIY STUDIES OF
RCMV ALL-03 IN RAT BRAIN ENDOTHELIAL CELLS
(RBEC)
4.1 Introduction 65
4.2 Materials and Methods 67
4.2.1 Coating of Cell Culture Surfaces 67
4.2.2 Sample Collection 67
4.2.3 Tissue Digestion 68
4.2.4 Purification of RBEC 69
4.2.5 Subcultivation of RBEC 71
4.2.6 Cryopreservation of RBEC 71
4.2.7 Viability Assessment of RBEC 72
4.2.8 Characterization of RBEC 73
4.2.8.1 Immunodetection of RBEC Markers 73
4.2.8.2 Polyacrylamide Gel Electrophoresis
(PAGE)
73
4.2.8.3 Scanning Electron Microscopy (SEM) 74
4.2.9 RCMV ALL-03 Infectivity Studies 75
4.2.9.1 Cytopathogenicity Studies 75
4.2.9.2 Virus Growth Curve 75
4.2.9.3 Haematoxylin and Eosin (H&E)
Staining
76
© COPYRIG
HT UPM
xiii
4.2.9.4 Transmission Electron Microscopy
(TEM)
77
4.3 Results 79
4.3.1 Brain Tissue Explants 79
4.3.2 RBEC Culture 80
4.3.3 Viability Assessment 82
4.3.4 Immunodetection of RBEC Markers 82
4.3.5 SDS-PAGE 84
4.3.6 SEM 85
4.3.7 Cytopathogenicity studies 86
4.3.8 Virus Growth Curve 88
4.3.9 H&E Staining 89
4.3.10 TEM 91
4.4 Discussion 92
4.5 Conclusions 96
5 HCMV SEROPREVALENCE IN MALAYSIA AND ITS
CROSS-REACTIVITY WITH RCMV ALL-03
5.1 Introduction 97
5.2 Materials and Methods 100
5.2.1 Sample Population 100
5.2.2 Serological Screening of HCMV 100
5.2.3 Type of Viruses 101
5.2.4 SDS-PAGE 102
5.2.4.1 Gel Components 102
5.2.4.2 Protein Quantification 103
5.2.4.3 Electrophoresis 103
5.2.4.4 Gel Staining 104
5.2.5 Western Blot 104
5.2.5.1 Protein Transfer to PVDF Membranes 104
5.2.5.2 Immunodetection of Blotted Proteins 106
5.2.6 Indirect Immunoperoxidase (IIP) 106
5.3 Results 108
5.3.1 ELISA 108
5.3.2 SDS-PAGE 109
5.3.3 Western Blot 112
5.3.4 IIP 112
5.4 Discussion 114
5.5 Conclusions
117
© COPYRIG
HT UPM
xiv
6 GENOME INFORMATICS OF RCMV ALL-03
AND ANALYSIS OF THE A033 GENE
6.1 Introduction 118
6.2 Materials and Methods 121
6.2.1 Cells and Virus Culture 121
6.2.2 Preparation of Genomic DNA 121
6.2.3 Determination of DNA Concentration and Purity 122
6.2.4 Agarose Gel Electrophoresis and Staining 122
6.2.5 Whole Genome Sequencing using NGS Illumina
Technology
123
6.2.6 NGS Analysis using CLC Genomics Workbench 124
6.2.6.1 Importing of Raw Data and Trimming
Reads
124
6.2.6.2 De novo Assembly 125
6.2.6.3 Reference Mapping 125
6.2.7 RCMV ALL-03 Draft Genome Analysis 128
6.2.7.1 Alignment of Contigs and Scaffolding 130
6.2.7.2 CMV Genome Comparison 130
6.2.7.3 Phylogenomics of CMV 131
6.2.7.4 Gene Prediction 131
6.2.7.5 Functional Gene Analysis 132
6.2.7.6 Annotation of the Draft Genome 132
6.2.8 A033 Gene Analysis 133
6.2.9 A033 Primer Design 133
6.2.10 A033 Primer Validation 134
6.2.10.1 In silico PCR 134
6.2.10.2 MFEprimer 134
6.2.10.3 SPCR 135
6.2.10.4 PCR 135
6.3 Results 137
6.3.1 Genomic DNA Extraction 137
6.3.2 DNA Concentration and Purity 137
6.3.3 Sequencing Data 137
6.3.4 De novo Assembly 138
6.3.5 Reference Mapping 140
6.3.6 Alignment of Contigs and Scaffolds 142
6.3.7 CMV Genome Comparisons 144
6.3.8 Phylogenomics 144
6.3.9 Genome Annotation and Gene Prediction 147
6.3.10 Gene Function Analysis 160
6.3.11 A033 Gene Analysis 162
6.3.12 A033 Primer Design 168
6.3.13 A033 Primer Validation 171
6.4 Discussion 172
6.5 Conclusions
179
© COPYRIG
HT UPM
xv
7 GENERAL DISCUSSION, CONCLUSIONS AND
RECOMMENDATIONS FOR FUTURE RESEARCH
7.1 General Discussion 180
7.2 Conclusions 186
7.3 Recommendations for Future Research 187
REFERENCES 188
APPENDICES
Appendix A : Media and Related Tissue Culture Solutions 214
Appendix B : TCID50 Calculation 217
Appendix C : Primary Cell Culture Media and Related Buffers 218
Appendix D : SEM and TEM Related Buffers and Solutions 220
Appendix E : H& E Staining Solutions 222
Appendix F : Supporting Evidence for RBEC SDS-PAGE 223
Appendix G : SDS-PAGE, Western Blot Buffers and Solutions 224
Appendix H : Standard Curve, ELISA and evidence for SDS-PAGE 228
Appendix I : Genome Informatics Analysis 232
Appendix J : Sequences of Annotated Genes in RCMV ALL-03 241
BIODATA OF STUDENT 275
LIST OF PUBLICATIONS 276